Defining the entire ensemble of molecular interactions and networks present in an organism (i.e., the Interactome) is essential for understanding the function, dynamics and logic underlying complex cellular systems. Large-scale efforts, involving both yeast-two-hybrid approaches and affinity-mass spectrometry, have begun to define large portions of the eukaryotic Interactome. While these approaches have proven effective for evaluating cytoplasmic interactions, they are inadequate for defining the interactions involving the 1/3 of the human proteome represented by secreted proteins and the ectodomains of integral membrane proteins. These proteins and their interactions are vital to cellular and multi-cellular processes as they detect the developmental, morphogenetic and environmental cues that are central to normal physiology and pathology. These receptor:ligand complexes also offer enormous promise as therapeutic targets and for the development of biologics to treat autoimmune diseases, infectious diseases and malignancies. At present there exists no platform to support the discovery of these extracellular interactions. To address this challenge, we have recently established strong proof-of-concept for two distinct high-throughput platforms for mapping of the ?Ecto-Interactome?, the entire set of interactions formed by secreted and cell surface proteins. These efforts exploit a multi-disciplinary team, composed of protein chemists, automation specialists and biologists, which is merging multiple protein expression/presentation strategies with cutting-edge cell microarray and flow cytometry technologies. The optimization and implementation of these platforms promises to have transformative impact by revealing extracellular interactions and networks that yield novel insights into normal physiology, disease and therapeutic strategies. We are positioned to make significant progress in assessing the feasibility of defining the Ecto-Interactome. Together with the considerable body of cytoplasmic interactions that is accruing, these studies will provide important insights into the full range of molecular circuitry that integrates multiple disparate signals into cellular and multi-cellular function.